CLPP exons and flanking intronic regions were PCR amplified using M13-tailed primers (Table S1 in Supplementary Material) and sequenced by the ABI 3730 automatic sequencer (Applied Biosystems, Foster City, CA, USA). CLPP Sanger sequencing was performed in all patients, sibs, and parents. Paternity was tested by multiplex VNTR analysis using the AmpFISTR Identifiler PCR amplification kit according to manufacturer’s manual (Applied Biosystems, Foster City, CA, USA).
Ampfistr identifiler pcr amplification kit
Manufactured by Thermo Fisher Scientific
Sourced in United States, Australia
The AmpFISTR Identifiler PCR Amplification Kit is a short tandem repeat (STR) multiplex assay designed for human identification. It amplifies 15 STR loci and the gender-determining locus, amelogenin, in a single PCR reaction. The kit provides a comprehensive set of reagents and protocols for efficient DNA amplification and analysis.
Automatically generated - may contain errors
Lab products found in correlation
Genemarker v1, by SoftGenetics (6 mentions)
Rpmi 1640 medium, by Lonza (4 mentions)
Fetal bovine serum (fbs), by Thermo Fisher Scientific (4 mentions)
Mycoalert, by Lonza (2 mentions)
Dmem f12 medium, by Lonza (2 mentions)
Dulbecco modified eagle medium (dmem), by Thermo Fisher Scientific (2 mentions)
Mycoalert mycoplasma detection kit, by Lonza (2 mentions)
Du145, by American Type Culture Collection (2 mentions)
Abi 3730, by Thermo Fisher Scientific (1 mentions)
Colo205, by American Type Culture Collection (1 mentions)
Crl 1831, by American Type Culture Collection (1 mentions)
Nsg b2m mice, by Jackson ImmunoResearch (1 mentions)
Rwpe 1, by American Type Culture Collection (1 mentions)
Lipofectamine 3000 transfection reagent, by Thermo Fisher Scientific (1 mentions)
Abi 3500 genetic analyzer, by Thermo Fisher Scientific (1 mentions)
Sk n mc, by Lonza (1 mentions)
Caski, by American Type Culture Collection (1 mentions)
Iscove s modified dulbecco s medium, by Lonza (1 mentions)
Collagenase, by Merck Group (1 mentions)
Complete dmem, by Lonza (1 mentions)
33 protocols using ampfistr identifiler pcr amplification kit
1
CLPP Gene Sequencing and Paternity Test
2
Colorectal Cancer Cell Lines and Tissue Samples
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The human normal colon epithelial cell line FHC (ATCC® CRL-1831™) and the human CRC cell lines HT-29 (ATCC® HTB-38™), SW480 (ATCC® CCL-228™) and COLO205 (ATCC® CCL-222™) were from American Type Culture Collection (ATCC). The human CRC cell line LIM1215 (ECACC 10092301) was from European Collection of Authenticated Cell Cultures (ECACC). Cells were cultured according to standard mammalian tissue culture protocols37 (link). Cells were cultured in a humidified incubator at 37 °C and 5% CO2 and were tested using RT-PCR for mycoplasma contamination. Cell lines were authenticated using the AmpFISTR Identifiler PCR Amplification Kit from Applied Biosystems and GeneMarker V1.91 software (SoftGenetics LLC). FFPE CRC tissues and paired normal colon tissues were retrieved from archives of the Anatomy Pathology Departments at Shanxi Cancer Hospital and Henan Provincial People’s Hospital. Freshly removed colon cancer and paired adjacent normal colon tissues were obtained from patients undergoing surgical resection at the Department of Colorectal Surgery at Shanxi Cancer Hospital. Studies using human tissues were approved by the Human Research Ethics Committees of Henan Provincial People’s Hospital and Shanxi Cancer Hospital in agreement with the guidelines set forth by the Declaration of Helsinki. All participants provided written informed consent.
3
Melanoma Cell Line Cultivation and Authentication
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Human melanoma cell lines cultured in DMEM containing 5% FCS has been described previously35 (link). Five paired melanoma tissue samples from patients pre- and post treatment with vemurafenib have been established and described previously20 (link). Melanoma cells selected for resistance to apoptosis induced by the mutant BRAF Inhibitor PLX4720 has been described previously9 (link). Individual cell line authentication has been confirmed using the AmpFISTRIdentifiler PCR Amplification Kit from Applied Biosystems (Mulgrave, VIC, Australia) and GeneMarker V1.91 software (SoftGenetics LLC, State College, PA) every 6 months. Cell lines were regularly tested for mycoplasma infection using Myco Alert according to the manufacturer’s protocol (Lonza, Walkersville, MD, USA). Studies using human tissues were approved by the Human Research Ethics Committees of the University of Newcastle and Royal Prince Alfred Hospital, Australia.
4
Colon Cancer Tissue Sourcing and Authentication
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ME4405, TRAIL.S, UMI-77.S, HCT116, P493-6, and HAFF cells were maintained in DMEM (Invitrogen) supplemented with 10% FBS and 1% penicillin/streptomycin. Cells were cultured in a humidified incubator at 37 °C and 5% CO2. All cell lines used tested negative for mycoplasma contamination and were subjected to authentication using the AmpFISTR Identifiler PCR Amplification Kit (Applied Biosystems) and GeneMarker V1.91 software (SoftGenetics LLC) (70 (link)). Formalin-fixed paraffin-embedded (FFPE) colon cancer tissues were obtained from the Department of Pathology at Shanxi Cancer Hospital and Institute (Taiyuan, China). Freshly removed colon cancer and paired adjacent noncancerous colon tissues were collected from patients undergoing surgical resection at the Department of General Surgery at Shanxi Cancer Hospital and Institute. Studies using human tissues were approved by the Human Research Ethics Committees of the University of Science and Technology of China and Shanxi Cancer Hospital and Institute in agreement with the guidelines set forth by the Declaration of Helsinki. The study is compliant with all relevant ethical regulations for human research participants, and all participants provided written informed consent.
5
Establishment of Leukemia PDX Models
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PDX models were generated as described previously [8 (link)] using a protocol approved by the Nemours Institutional Animal Care and Use Committee. Leukemic cells from patient samples were injected into immune-deficient NSG-B2m mice (stock no. 010636, Jackson Laboratories, Bar Harbor, ME, USA) via the tail vein. Disease progression was examined by determination of the percentage of human leukemic cells in mouse peripheral blood by flow cytometry. Mice were closely monitored for experimental endpoints such as increased leukemic burden, weight loss greater than 20% body weight, hunched back, and impaired mobility. Mice meeting end point criteria were euthanized using a method consistent with the guidelines of the American Veterinary Medical Association. Leukemic cells were isolated from the femurs and spleen post euthanasia and used for serial transplantation in a new cohort of mice. Bioauthentication and validation of PDX sample with matching primary sample was performed by subjecting the DNA samples to AmpFISTR Identifiler PCR Amplification Kit (Applied Biosystems, Foster City, CA, USA).
6
Establishment and Characterization of DMPM Cell Lines
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Human DMPM cell lines (STO and MesoII) were established from surgical specimens of patients who underwent surgery at Fondazione IRCCS Istituto Nazionale dei Tumori of Milan, as previously described [6 (link)]. The normal human lung fibroblast (WI38) and the normal adult human prostate (RWPE-1) cell lines were obtained from the American Type Culture Collection (ATCC; #CCL-75 and #CRL-11609). Cells were maintained in the logarithmic growth phase as a monolayer in DMEM F12 (STO and MesoII) and DMEM (WI38) media (Lonza; #12-719F and #12-604F) supplemented with 10% heat-inactivated fetal bovine serum, or in K-SFM (RWPE-1; GIBCO; #17005-042), in a humidified incubator at 37°C with a supply of 5% CO2/95% air atmosphere. Cell lines are tested fortnightly for the absence of Mycoplasma and periodically (every six months) monitored for DNA profile of short tandem repeats analysis by the AmpFISTR Identifiler PCR amplification kit (Applied Biosystems; #4322288). All cell lines were last tested in September 2014.
7
Cell Line Characterization and Manipulation
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PDAC and TNBC cells were obtained from American Type Culture Collection (ATCC) and BXPC3 vector control, RON knockdown clones were provided by Dr. Freeman.14 Cell line identities were verified at TGEN by short tandem repeat (STR) profiling using the AmpFISTR Identifiler PCR Amplification Kit (Applied Biosystems). Results were compared with published STR sequences from the ATCC. The STR profiling is repeated once a cell line has been passaged more than 6 months after previous STR profiling. Transient knockdown of RON in CFPAC1 cells was done using siRNA RON (SC‐36434) and HIF‐1α knockdown in BXPC3 cells using siHIF‐1α (SC‐44225) and Lipofectamine 3000 transfection reagent (Invitrogen). Panc1 vector and Panc1 FL‐RON cells were generated through a stable expression of CMV‐Neo and CMV‐FL‐RON (full‐length RON cDNA) plasmids, respectively. Human recombinant MSP was purchased from R&D Systems and RON TKI, LCRF‐0004 (will be referred to as LCRF) was provided by Dr. Raeppel.41
8
Paternity Verification in Fetal Cases
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To confirm paternity results in alleged family cases, conventional STR‐based paternity testing using fetal genomic DNA extracted from amniotic fluid or buccal swabs was performed. The AmpFISTR Identifiler PCR amplification kit (Applied Biosystems) was employed, following the manufacturer's protocol and according to strict AABB standards. For alleged family cases with male fetus where amniotic fluid was not available, cross‐validation of the paternity results was performed through additional testing with maternal cfDNA using the AmpFISTR Yfiler PCR amplification kit (Applied Biosystems), following the manufacturer's protocol modified for use with cfDNA. Parental genomic DNA was analyzed in parallel in each case. Capillary electrophoresis (50‐cm capillary array, POP‐7) of PCR amplicons from both kits were conducted in an ABI 3500 Genetic Analyzer (Applied Biosystems) according to the manufacturer's instructions and strict AABB standards, and data were analyzed using the GeneMapper ID software v.5 (Applied Biosystems). The associated paternity probabilities were calculated according to equations similar to those described above but based on frequencies of the matched STRs in the population of the alleged father.
9
Cell Line Authentication and Culture
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We used four human SCC cell lines, CaSki and SiHa, from uterine cervix and A431, from skin, along with the TPT-resistant subline (A431/TPT) selected in our lab [24 (link)], two human Ewing's sarcoma family of tumor cell lines SK-N-MC (Askin's tumor) and TC71 (Ewing's sarcoma) and the human embryonal rhabdomyosarcoma cell line RD. All SCC, SK-N-MC and RD cell lines were cultured in RPMI-1640 medium (Lonza, Verviers, Belgium), whereas the TC71 cell line was maintained in Iscove's modified Dulbecco's Medium (Lonza). All cell lines were grown in medium supplemented with 10% FBS and maintained in a humidified incubator with 5% CO2 at 37°C. The A431, CaSki and SiHa cell lines were obtained from American Type Culture Collection (Manassas, VA). The SK-N-MC cell line was kindly provided by R. Maggi (University of Milan, Italy), the RD cell line by A. Rosolen (University of Padua, Italy), and the TC71 cell line by M.C. Manara (Rizzoli Institute, Bologna, Italy). Cell lines were authenticated by single tandem repeat analysis by the AmpFISTR Identifiler PCR amplification kit (Applied Biosystems, Foster City, CA).
10
Establishment of Patient-Derived Xenograft and Cell Line
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A fresh tumor specimen was collected at the time of the surgical resection, aseptically dissected, cut into small fragments (3 × 3 × 3 mm) and grafted subcutaneously into the right flank of 6–8-week-old female SCID mice. Tumor growth was followed by biweekly measurement of tumor diameters with a Vernier calliper. Tumor volume (TV) was calculated according to the formula: TV (mm3) = d2 × D/2, where d and D are the shortest and the longest diameters, respectively. After the third passage in mice, and based on growth characteristics, the PDX was considered established.
To generate the corresponding cell line, the PDX was enzymatically digested with collagenase (200 U/mL) (Sigma, St. Louis, MO, USA) for 3 h at 37 °C. collagenase was inactivated by two washes in fetal bovine serum (FBS). Cells were then resuspended in complete DMEM (Lonza, Verviers, Belgium). When cells reached 70–80% confluence, they were propagated under optimal culture conditions. The origins of both PDX and the cell line were authenticated through microsatellite analysis by the AmpFISTR Identifiler PCR amplification kit (Applied Biosystems, PN4322288, Foster City, CA, USA).
To generate the corresponding cell line, the PDX was enzymatically digested with collagenase (200 U/mL) (Sigma, St. Louis, MO, USA) for 3 h at 37 °C. collagenase was inactivated by two washes in fetal bovine serum (FBS). Cells were then resuspended in complete DMEM (Lonza, Verviers, Belgium). When cells reached 70–80% confluence, they were propagated under optimal culture conditions. The origins of both PDX and the cell line were authenticated through microsatellite analysis by the AmpFISTR Identifiler PCR amplification kit (Applied Biosystems, PN4322288, Foster City, CA, USA).
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